Showing papers by "Joseph S. Fruton published in 1971"

The comparative specificity of acid proteinases.

Abstract: Examination of the kinetic parameters for the hydrolysis, by acid proteinases, of a single peptide bond (between p-nitro-L-phenylalanyl and L-phenylalanyl) in a series of oligopeptides has shown that secondary interactions are important factors in determining the catalytic efficiency. Comparison of the action of highly purified pepsinlike enzymes (Rhizopus proteinase, Mucor proteinase, rennin) with that of swine pepsin A indicates significant differences among them, either in the binding of the substrate (as estimated by Km), or in the catalytic efficiency (as measured by kcat), or both. It may be concluded from these data that, in their action on oligopeptide substrates, the specificity of proteinases operating by a similar catalytic mechanism cannot be explained solely in terms of the amino acid residues flanking the sensitive peptide bond; in addition, the specificity includes significant contributions from secondary interactions arising from complementary relations between parts of the substrate and of the enzyme at a distance from the catalytic site. Data are also presented for the effect of urea (about 1 M) on the kinetic parameters of several acid proteinases; under the conditions of these studies, the binding of the substrate is affected to a much lesser degree than is the catalytic efficiency.

Bifunctional inhibitors of pepsin.

Abstract: Two bifunctional reagents designed to probe the active site of pepsin and other acid proteinases are described. One of these, the bisdiazoketone 1,1-bis(diazoacetyl)-2-phenylethane inactivates pepsin at pH 5.0 much more rapidly than the corresponding monodiazoketon 1-diazoacetyl-2-phenylethane, whereas the other, the bromodiazoketone dl-1-diazoacetyl-1-bromo-2-phenylethane is less effective in this regard. The inactivation is greatly accelerated by the presence of Cu(II), and the pH dependence of the process is consistent with the interaction of the enzyme with the metal complex of the carbene derived from the reagent. The bisdiazoketone appears to react stoichiometrically with pepsin in a 1:1 ratio to form a product whose apparent molecular size is the same as that of untreated pepsin. The inactivation of pepsin by the bromodiazoketone is accompanied by the release of stoichiometric amounts of bromide ions and the formation of a major product whose apparent size is similar to that of pepsin, and a minor component larger than the untreated enzyme.